Gene expression and chromatin conformation of microglia in virally suppressed people with HIV

Leveraging our rapid autopsy “Last Gift” cohort at UCSD, we identified distinct microglial gene expression profiles despite ART.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): Given that the brain is a putative reservoir for HIV-1, the authors leveraged the Last Gift Cohort to investigate variation of microglia in the DLPFC based on transcriptomic signatures and the chromatin accessibility landscape to inform and potentially correlate gene expression data that on open and hence, transcriptionally-active regions.The authors do not overstate the conclusions of this observational study conducted on three male brains from individuals with very different co-morbid conditions.Namely, they identified 15 different CD45+ clusters and based on a limited set of myeloid specific genes attempted to parse out the six microglia clusters, from perivascular macrophages, monocytes and neutrophils.Alignment of sequence data with a reference HIV genome was used to identify cells harboring HIV DNA and RNA.Previous relevant studies are cited and this study adds to the field and shows what insights are possible using human brain tissue and the significant limitations to be overcome in future experimental designs.The contributions of all the authors to the study as presented is not clear.
Reviewer #2 (Comments to the Authors (Required)): In "Gene expression profiles and chromatin conformation of microglia in virally suppressed people with HIV" , Schlachetzki et al.Isolated viable microglia from post-mortem brain tissue of 3 donors with HIV that were on antiviral combination therapy until shortly before they passed away.They then subjected these cells to single-cell RNA sequencing as well as scATACseq to detect any possible presence of HIV virus, either expressed as RNA, or integrated in the genome.Using the scRNA data they further analyzed the effects of HIV on the expression pattern of infected microglia.The number of replicates in the dataset is understandably low, with data available from 3 donors.Overall, the quality of the data seems high and given the unique nature of the tissue, these data still represents a valuable resource.There are a few points that could use some clarification prior to publishing: 1st Authors' Response to Reviewers July 4, 2024 Reviewer #1 (Comments to the Authors (Required)): Given that the brain is a putative reservoir for HIV-1, the authors leveraged the Last Gift Cohort to investigate variation of microglia in the DLPFC based on transcriptomic signatures and the chromatin accessibility landscape to inform and potentially correlate gene expression data that on open and hence, transcriptionally-active regions.The authors do not overstate the conclusions of this observational study conducted on three male brains from individuals with very different co-morbid conditions.Namely, they identified 15 different CD45+ clusters and based on a limited set of myeloid specific genes attempted to parse out the six microglia clusters, from perivascular macrophages, monocytes and neutrophils.Alignment of sequence data with a reference HIV genome was used to identify cells harboring HIV DNA and RNA.Previous relevant studies are cited and this study adds to the field and shows what insights are possible using human brain tissue and the significant limitations to be overcome in future experimental designs.
Response: We would like to thank Reviewer 1 for the positive comments on our manuscript.
The contributions of all the authors to the study as presented is not clear.
Response: We now added the following paragraph to the Acknowledgment section outlining the contributions of all the authors to the study.
"JCMS conceived and designed the study, processed tissue to isolate microglia, analyzed multi-omics data, interpreted the results, wrote the first draft of the manuscript.SG co-directs the Last Gift Program and performed all rapid autopsies with her team, interpreted the data, edited the manuscript.ZO processed scRNA-seq data and performed quality control of all single cell data.AJL collected samples, performed microglia isolation and library preparations, edited the manuscript.XY interpreted the single cell RNA-seq data, edited the manuscript.SMO performed library preparations, edited the manuscript.JFC processed scATAC-seq data.PJG interpreted the data, edited the manuscript.KLJS interpreted the data, edited the manuscript.AC helped with rapid autopsy procedures analyzed multi-omics data, edited the manuscript.DM collected clinical data, edited the manuscript.CLA interpreted the data, edited the manuscript.RJE interpreted the data, edited the manuscript.DMS co-directs of the Last Gift Program, helped with rapid autopsy procedures, interpreted the data, edited the manuscript.CKG supervised the project, interpreted the data, edited the manuscript." Reviewer #2 (Comments to the Authors (Required)): In "Gene expression profiles and chromatin conformation of microglia in virally suppressed people with HIV" , Schlachetzki et al.Isolated viable microglia from postmortem brain tissue of 3 donors with HIV that were on antiviral combination therapy until shortly before they passed away.They then subjected these cells to single-cell RNA sequencing as well as scATACseq to detect any possible presence of HIV virus, either expressed as RNA, or integrated in the genome.Using the scRNA data they further analyzed the effects of HIV on the expression pattern of infected microglia.The number of replicates in the dataset is understandably low, with data available from 3 donors.Overall, the quality of the data seems high and given the unique nature of the tissue, these data still represents a valuable resource.
Response: We would like to thank Reviewer 2 for appreciating our work.
There are a few points that could use some clarification prior to publishing: 1.In figure 4 you analyse the gene expression differences of the microglia cells that are positive for HIV contrasted to a random selection of microglia cells from the population.The majority of the HIV positive cells are assigned to clusters that are not homeostatic microglia but overall express markers of cellular stress, inflammation or activation.Therefore, the fact that these gene expression differences are found could merely reflect the expression signature of that cluster, rather then an expression pattern induced by HIV.In these clusters, there are many cells with a similar pattern that are not HIV positive.So are the genes induced by hIV?Or are the cells that show this pattern more likely to be infected?And are there any gene expression changes when you compare HIV positive and negative cells from the same cluster?
Response: We agree that the majority of HIV-positive cells reside in clusters that are not representative of homeostatic microglia.This raises the possibility that the observed differences may reflect the general activation state of these clusters rather than a direct effect of HIV infection.As suggested by the reviewer, we reanalyzed our data and compared HIV RNA positive and negative cells from the same cluster.We found that the gene expression profiles of microglia with detectable HIV RNA are very similar to those of microglia with no detectable HIV RNA (Fig. 1 for reviewers).Specifically, we performed differential gene expression analysis of HIV RNA+ microglia (Cluster 1: 19 cells, Cluster 3: 19 cells, Cluster 4: 26 cells, Cluster 7: 14 cells) and compared them to randomly selected similar cell numbers from the same cluster.No significant changes in gene expression were found in Clusters 3 and 7, and only 6 and 1 genes were significantly altered in Clusters 1 and 4, respectively.
2. In the discussion on page 11 you write a section on defective-proviruses being able to transcribe HIV RNA.Then how you detect HIV RNA in 0.005% of microglia that you go on to assign to coming from replication-competent provirus.How do you know what type of virus (defective or replication-competent) was present in these microglia?
Response: The reviewer is correct; we cannot distinguish whether the HIV RNA detected in microglia originates from defective or replication-competent proviruses.Both types of viruses can contribute to the immune response, but our current methods do not allow us to differentiate between them in this context.We recognize the importance of this distinction and its implications for understanding the role of HIV-infected microglia in the immune response and viral persistence.However, due to the limitations of our current detection techniques, we are unable to address this differentiation in our study.Future research utilizing more advanced methodologies may be able to provide clearer insights into the specific contributions of defective versus replication-competent HIV in microglia.We now removed this section from the discussion.
3. In the discussion it is written that: Additional markers, e.g., H3K27ac for active promoters and enhancers...The methods section, which includes headings: Nuclei isolation from frozen tissue, FANS sorting of PU.1+ nuclei and H3K27ac ChIPseq on  competent HIV viruses, we have removed these sentences from the discussion on page 11 to provide a more succinct and clear discussion.Removed sentences from the manuscript: Although effective ART prevents the full replication cycle, current ART regimens do not entirely prevent the transcription of HIV genes.It is estimated that roughly 25-30% -including those harboring defective proviruses-transcribe HIV RNA even during suppressive ART.Thank you for submitting your revised manuscript entitled "Gene expression and chromatin conformation of microglia in virally suppressed people with HIV".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Fig. 1 :
Fig. 1: Differential gene expression analysis within in clusters between HIV-RNA+ microglia and microglia without detectable HIV-RNA.
Science Alliance Manuscript #LSA-2024-02736-TR Dr. Christopher K Glass University of California, San Diego Division Cell and Molecular Medicine University of California -San Diego 9500 Gilman Dr La Jolla, CA 92093-0651 Dear Dr. Glass, and S1A-C to your main manuscript text.Each section of each figure needs to be called out -please indicate that written informed consent was obtained from the patients If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date. http://www.lsajournal.org Thank you for submitting your Resource entitled "Gene expression and chromatin conformation of microglia in virally suppressed people with HIV".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.